Polynucleotide encoding mammalian cytokine-like polypeptide-10

ABSTRACT

A mammalian cytokine-like polypeptide, called Mammalian Cytokine-like polypeptide-10, (Zcyto10), polynucleotides encoding the same, antibodies which specifically bind to the polypeptide, and anti-idiotypic antibodies which bind to the antibodies. Zcyto10 is useful for promoting the healing of wounds and for stimulating the proliferation of platelets.

The present application is a continuation of U.S. patent applicationSer. No. 11/458,910, filed Jul. 20, 2006, now abandoned which is adivisional of U.S. patent application Ser. No. 10/789,129, filed Feb.27, 2004, now U.S. Pat. No. 7,119,191, which is a continuation of U.S.patent application Ser. No. 10/413,661, filed Apr. 15, 2003, now U.S.Pat. No. 7,115,714, which is a continuation of U.S. patent applicationSer. No. 09/313,458, filed May 17, 1999, now U.S. Pat. No. 6,576,743,which is a continuation-in-part of U.S. patent application Ser. No.09/199,586, filed Nov. 25, 1998, now abandoned which claims the benefitof U.S. Provisional Application No. 60/066,597, filed Nov. 26, 1997, allof which are herein incorporated by reference.

BACKGROUND OF THE INVENTION

Proliferation and differentiation of cells of multicellular organismsare controlled by hormones and polypeptide growth factors. Thesediffusable molecules allow cells to communicate with each other and actin concert to form cells and organs, and to repair and regeneratedamaged tissue. Examples of hormones and growth factors include thesteroid hormones (e.g. estrogen, testosterone), parathyroid hormone,follicle stimulating hormone, the interleukins, platelet derived growthfactor (PDGF), epidermal growth factor (EGF), granulocyte-macrophagecolony stimulating factor (GM-CSF), erythropoietin (EPO) and calcitonin.

Hormones and growth factors influence cellular metabolism by binding toproteins. Proteins may be integral membrane proteins that are linked tosignaling pathways within the cell, such as second messenger systems.Other classes of proteins are soluble molecules.

Of particular interest are cytokines, molecules that promote theproliferation and/or differentiation of cells. Examples of cytokinesinclude erythropoietin (EPO), which stimulates the development of redblood cells; thrombopoietin (TPO), which stimulates development of cellsof the megakaryocyte lineage; and granulocyte-colony stimulating factor(G-CSF), which stimulates development of neutrophils. These cytokinesare useful in restoring normal blood cell levels in patients sufferingfrom anemia or receiving chemotherapy for cancer. The demonstrated invivo activities of these cytokines illustrates the enormous clinicalpotential of, and need for, other cytokines, cytokine agonists, andcytokine antagonists.

SUMMARY OF THE INVENTION

The present invention addresses this need by providing a novelpolypeptide and related compositions and methods. Within one aspect, thepresent invention provides an isolated polynucleotide encoding amammalian four alpha helix cytokine termed Zcyto10. The human Zcyto10polypeptide is comprised of a sequence of 176 amino acids with theinitial Met as shown in SEQ ID NO:1 and SEQ ID NO:2. It is believed thatamino residues 1-24 are signal sequence, and the mature Zcyto10polypeptide is represented by the amino acid sequence comprised ofresidues 25, a leucine, through amino acid residue 176, a glutamic acidresidue, also defined by SEQ ID NO:12. Another embodiment of the presentinvention is defined by the sequences of SEQ ID NO: 3 and SEQ ID NO: 4.The polypeptide of SEQ ID NO: 4 is comprised of 151 amino acid residueswherein amino acids 1-24 comprise a signal sequence and the maturesequence is comprised of amino acid residues 25, a leucine, throughamino acid 151 a glutamic acid, also defined by SEQ ID NO:13. Anotheractive variant is comprised of amino acid residues 33, a cysteine,through amino acid residue 176 of SEQ ID NO:2. This variant is alsodefined by SEQ ID NO:26.

Mouse Zcyto10 is also a polypeptide comprised of 176 amino acid residuesas defined by SEQ ID NOs: 18 and 19. Mouse Zcyto10 has a signal sequenceextending from amino acid residue 1, a methionine, extending to andincluding amino acid residue 24, a glycine of SEQ ID NO:19. Thus, themature mouse Zcyto10 extends from amino acid residue 25, a leucine, toand including amino acid residue 176 a leucine of SEQ ID NO:19, alsodefined by SEQ ID NO:20. Another active variant is believed to extendfrom amino acid 33, a cysteine, through amino acid 176, of SEQ ID NO:19.This variant is also defined by SEQ ID NO:25. Within an additionalembodiment, the polypeptide further comprises an affinity tag.

A variant of mouse Zcyto10 is defined by SEQ ID NOs: 33 and 34. Thisvariant is 154 amino acid residues in length and has a signal sequenceextending from amino acid residue 1, a methionine, to and includingamino acid residue 24, a glycine, of SEQ ID NO:34. Thus, the maturesequence extends from amino acid residue 25, a leucine, to and includingamino acid residue 154, a leucine, of SEQ ID NO:34. The mature sequenceis also defined by SEQ ID NO:35.

Within a second aspect of the invention there is provided an expressionvector comprising (a) a transcription promoter; (b) a DNA segmentencoding Zcyto10 polypeptide, and (c) a transcription terminator,wherein the promoter, DNA segment, and terminator are operably linked.

Within a third aspect of the invention there is provided a culturedeukaryotic or prokaryotic cell into which has been introduced anexpression vector as disclosed above, wherein said cell expresses apolypeptide encoded by the DNA segment.

Within a further aspect of the invention there is provided a chimericpolypeptide consisting essentially of a first portion and a secondportion joined by a peptide bond. The first portion of the chimericpolypeptide consists essentially of (a) a Zcyto10 polypeptide as shownin SEQ ID NO: 2 (b) allelic variants of SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:25, SEQ IDNO:26 SEQ ID NO:34 or SEQ ID NO:35; and (c) protein polypeptides thatare at least 90% identical to (a) or (b). The second portion of thechimeric polypeptide consists essentially of another polypeptide such asan affinity tag. Within one embodiment the affinity tag is animmunoglobulin F_(c) polypeptide. The invention also provides expressionvectors encoding the chimeric polypeptides and host cells transfected toproduce the chimeric polypeptides.

Within an additional aspect of the invention there is provided anantibody that specifically binds to a Zcyto10 polypeptide as disclosedabove, and also an anti-idiotypic antibody which neutralizes theantibody to a Zcyto10 polypeptide.

Within another aspect of the present invention there is provided apharmaceutical composition comprising purified Zcyto10 polypeptide incombination with a pharmaceutically acceptable vehicle. Suchcompositions may be useful for modulating of cell proliferation, celldifferentiation or cytokine production in the prevention or treatment ofconditions characterized by improper cell proliferation, celldifferentiation or cytokine production, as are further discussed herein.More specifically, Zcyto10 polypeptide may be useful in the preventionor treatment of autoimmune diseases by inhibiting a cellular immuneresponse. Autoimmune diseases which may be amenable to Zcyto10 treatmentinclude IDDM, multiple sclerosis, rheumatoid arthritis and the like.Also, Zcyto10 polypeptides of the present invention may be useful ininhibiting cancer cell growth or proliferation.

Zcyto10 polypeptides of the present invention may also stimulate theimmune system to better combat microbial or viral infections. Inparticular, Zcyto10 can be administered systemically to increaseplatelet production by an individual. Moreover, Zcyto10 polypeptides ofthe present invention may be used in trachea-specific ortracheobronchial-specific applications, such as in the maintenance orwound repair of the tracheobronchial epithelium or cells underlying thesame, in regulating mucous production or mucocilary clearance of debrisor in treatment of asthma, bronchitis or other diseases of thetracheobronchial tract. It may also enhance wound healing and promoteregeneration of affected tissues which may be especially useful in thetreatment of periodontal disease. Furthermore, Zcyto10 polypeptides canbe used to treat skin conditions such as psoriasis, eczema and dry skinin general.

An additional embodiment of the present invention relates to a peptideor polypeptide which has the amino acid sequence of an epitope-bearingportion of a Zcyto10 polypeptide having an amino acid sequence describedabove. Peptides or polypeptides having the amino acid sequence of anepitope-bearing portion of a Zcyto10 polypeptide of the presentinvention include portions of such polypeptides with at least nine,preferably at least 15 and more preferably at least 30 to 50 aminoacids, although epitope-bearing polypeptides of any length up to andincluding the entire amino acid sequence of a polypeptide of the presentinvention described above are also included in the present invention.Also claimed are any of these polypeptides that are fused to anotherpolypeptide or carrier molecule. Such epitope variants include but arenot limited to SEQ ID NOs: 25-32. Antibodies produced from theseepitope-bearing portions of Zcyto10 can be used in purifying Zcyto10from cell culture medium.

These and other aspects of the invention will become evident uponreference to the following detailed description and the attacheddrawing.

DETAILED DESCRIPTION OF THE INVENTION

The teachings of all the references cited herein are incorporated intheir entirety by reference.

Prior to setting forth the invention in detail, it may be helpful to theunderstanding thereof to define the following terms:

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A, Nilsson et al., EMBO J. 4:1075 (1985);Nilsson et al., Methods Enzymol. 198:3 (1991), glutathione Stransferase, Smith and Johnson, Gene 67:31 (1988), Glu-Glu affinity tag,Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4 (1985),substance P, Flag™ peptide, Hopp et al., Biotechnology 6:1204-1210(1988), streptavidin binding peptide, or other antigenic epitope orbinding domain. See, in general, Ford et al., Protein Expression andPurification 2: 95-107 (1991). DNAs encoding affinity tags are availablefrom commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).

The term “allelic variant” is used herein to denote any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity of<10⁹ M⁻¹.

The term “complements of a polynucleotide molecule” is a polynucleotidemolecule having a complementary base sequence and reverse orientation ascompared to a reference sequence. For example, the sequence 5′ ATGCACGGG3′ is complementary to 5′ CCCGTGCAT 3′.

The term “contig” denotes a polynucleotide that has a contiguous stretchof identical or complementary sequence to another polynucleotide.Contiguous sequences are said to “overlap” a given stretch ofpolynucleotide sequence either in their entirety or along a partialstretch of the polynucleotide. For example, representative contigs tothe polynucleotide sequence 5′-ATGGCTTAGCTT-3′ are 5′-TAGCTTgagtct-3′and 3′-gtcgacTACCGA-5′.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

The term “expression vector” is used to denote a DNA molecule, linear orcircular, that comprises a segment encoding a polypeptide of interestoperably linked to additional segments that provide for itstranscription. Such additional segments include promoter and terminatorsequences, and may also include one or more origins of replication, oneor more selectable markers, an enhancer, a polyadenylation signal, etc.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both.

The term “isolated”, when applied to a polynucleotide, denotes that thepolynucleotide has been removed from its natural genetic milieu and isthus free of other extraneous or unwanted coding sequences, and is in aform suitable for use within genetically engineered protein productionsystems. Such isolated molecules are those that are separated from theirnatural environment and include cDNA and genomic clones. Isolated DNAmolecules of the present invention are free of other genes with whichthey are ordinarily associated, but may include naturally occurring 5′and 3′ untranslated regions such as promoters and terminators. Theidentification of associated regions will be evident to one of ordinaryskill in the art (see for example, Dynan and Tijan, Nature 316:774-78(1985).

An “isolated” polypeptide or protein is a polypeptide or protein that isfound in a condition other than its native environment, such as apartfrom blood and animal tissue. In a preferred form, the isolatedpolypeptide is substantially free of other polypeptides, particularlyother polypeptides of animal origin. It is preferred to provide thepolypeptides in a highly purified form, i.e. greater than 95% pure, morepreferably greater than 99% pure. When used in this context, the term“isolated” does not exclude the presence of the same polypeptide inalternative physical forms, such as dimers or alternatively glycosylatedor derivatized forms.

The term “operably linked”, when referring to DNA segments, indicatesthat the segments are arranged so that they function in concert fortheir intended purposes, e.g., transcription initiates in the promoterand proceeds through the coding segment to the terminator.

The term “ortholog” denotes a polypeptide or protein obtained from onespecies that is the functional counterpart of a polypeptide or proteinfrom a different species. Sequence differences among orthologs are theresult of speciation.

“Paralogs” are distinct but structurally related proteins made by anorganism. Paralogs are believed to arise through gene duplication. Forexample, a-globin, b-globin, and myoglobin are paralogs of each other.

A “polynucleotide” is a single- or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules. Sizes of polynucleotides are expressedas base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases(“kb”). Where the context allows, the latter two terms may describepolynucleotides that are single-stranded or double-stranded. When theterm is applied to double-stranded molecules it is used to denoteoverall length and will be understood to be equivalent to the term “basepairs”. It will be recognized by those skilled in the art that the twostrands of a double-stranded polynucleotide may differ slightly inlength and that the ends thereof may be staggered as a result ofenzymatic cleavage; thus all nucleotides within a double-strandedpolynucleotide molecule may not be paired. Such unpaired ends will ingeneral not exceed 20 nt in length.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides”.

The term “promoter” is used herein for its art-recognized meaning todenote a portion of a gene containing DNA sequences that provide for thebinding of RNA polymerase and initiation of transcription. Promotersequences are commonly, but not always, found in the 5′ non-codingregions of genes.

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

The term “receptor” denotes a cell-associated protein that binds to abioactive molecule (i.e., a ligand) and mediates the effect of theligand on the cell. Membrane-bound receptors are characterized by amulti-domain structure comprising an extracellular ligand-binding domainand an intracellular effector domain that is typically involved insignal transduction. Binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell. This interactionin turn leads to an alteration in the metabolism of the cell. Metabolicevents that are linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids. In general, receptors can be membranebound, cytosolic or nuclear; monomeric (e.g., thyroid stimulatinghormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGFreceptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSFreceptor, erythropoietin receptor and IL-6 receptor).

The term “secretory signal sequence” denotes a DNA sequence that encodesa polypeptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger polypeptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

The term “splice variant” is used herein to denote alternative forms ofRNA transcribed from a gene. Splice variation arises naturally throughuse of alternative splicing sites within a transcribed RNA molecule, orless commonly between separately transcribed RNA molecules, and mayresult in several mRNAs transcribed from the same gene. Splice variantsmay encode polypeptides having altered amino acid sequence. The termsplice variant is also used herein to denote a protein encoded by asplice variant of an mRNA transcribed from a gene.

Molecular weights and lengths of polymers determined by impreciseanalytical methods (e.g., gel electrophoresis) will be understood to beapproximate values. When such a value is expressed as “about” X or“approximately” X, the stated value of X will be understood to beaccurate to ±10%.

It is believed that Zcyto10 is of a member of the IL-10 subfamily ofcytokines. Other members of this group include MDA-7, IL-19, and KFF.Conserved amino acids in the helix D of Zcyto10 can be used as a tool toidentify new family members. Helix D is the most highly conserved havingabout 32% identity with the helix D of IL-10. For instance, reversetranscription-polymerase chain reaction (RT-PCR) can be used to amplifysequences encoding the conserved [the domain, region or motif fromabove] from RNA obtained from a variety of tissue sources or cell lines.In particular, highly degenerate primers designed from the Zcyto10sequences are useful for this purpose.

Within preferred embodiments of the invention the isolatedpolynucleotides will hybridize to similar sized regions of SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:18, SEQ ID NO:33 or a sequence complementarythereto, under stringent conditions. In general, stringent conditionsare selected to be about 5° C. lower than the thermal melting point(T_(m)) for the specific sequence at a defined ionic strength and pH.The T_(m) is the temperature (under defined ionic strength and pH) atwhich 50% of the target sequence hybridizes to a perfectly matchedprobe. Typical stringent conditions are those in which the saltconcentration is about 0.02 M or less at pH 7 and the temperature is atleast about 60° C. As previously noted, the isolated polynucleotides ofthe present invention include DNA and RNA. Methods for isolating DNA andRNA are well known in the art. Total RNA can be prepared using guanidineHCl extraction followed by isolation by centrifugation in a CsClgradient [Chirgwin et al., Biochemistry 18:52-94, (1979)]. Poly (A)⁺ RNAis prepared from total RNA using the method of Aviv and Leder, Proc.Natl. Acad. Sci. USA 69:1408-1412 (1972). Complementary DNA (cDNA) isprepared from poly(A)⁺ RNA using known methods. Polynucleotides encodingZcyto10 polypeptides are then identified and isolated by, for example,hybridization or PCR.

Additionally, the polynucleotides of the present invention can besynthesized using a DNA synthesizer. Currently the method of choice isthe phosphoramidite method. If chemically synthesized double strandedDNA is required for an application such as the synthesis of a gene or agene fragment, then each complementary strand is made separately. Theproduction of short genes (60 to 80 bp) is technically straightforwardand can be accomplished by synthesizing the complementary strands andthen annealing them. For the production of longer genes (>300 bp),however, special strategies must be invoked, because the couplingefficiency of each cycle during chemical DNA synthesis is seldom 100%.To overcome this problem, synthetic genes (double-stranded) areassembled in modular form from single-stranded fragments that are from20 to 100 nucleotides in length. See Glick, Bernard R. and Jack J.Pasternak, Molecular Biotechnology, Principles & Applications ofRecombinant DNA, (ASM Press, Washington, D.C. 1994), Itakura, K. et al.Synthesis and use of synthetic oligonucleotides. Annu. Rev. Biochem. 53:323-356 (1984), and Climie, S. et al. Chemical synthesis of thethymidylate synthase gene. Proc. Natl. Acad. Sci. USA 87:633-637 (1990).

Those skilled in the art will recognize that the sequences disclosed inSEQ ID NOs:1, 2, 3 and 4 represent a two alleles of the human, and SEQID NOs:18, 19, 33 and 34 represent two alleles of the mouse. Additionalallelic variants of these sequences can be cloned by probing cDNA orgenomic libraries from different individuals according to standardprocedures. Allelic variants of this sequence can be cloned by probingcDNA or genomic libraries from different individuals according tostandard procedures. Allelic variants of the DNA sequence shown in SEQID NO:1, including those containing silent mutations and those in whichmutations result in amino acid sequence changes, are within the scope ofthe present invention, as are proteins which are allelic variants of SEQID NO:2. cDNAs generated from alternatively spliced mRNAs, which retainthe properties of the Zcyto10 polypeptide are included within the scopeof the present invention, as are polypeptides encoded by such cDNAs andmRNAs. Allelic variants and splice variants of these sequences can becloned by probing cDNA or genomic libraries from different individualsor tissues according to standard procedures known in the art.

The present invention further provides counterpart proteins andpolynucleotides from other species (“species orthologs”). Of particularinterest are Zcyto10 polypeptides from other mammalian species,including murine, porcine, ovine, bovine, canine, feline, equine, andother primates. Species orthologs of the human Zcyto10 protein can becloned using information and compositions provided by the presentinvention in combination with conventional cloning techniques. Forexample, a cDNA can be cloned using mRNA obtained from a tissue or celltype that expresses the protein. Suitable sources of mRNA can beidentified by probing Northern blots with probes designed from thesequences disclosed herein. A library is then prepared from mRNA of apositive tissue or cell line. A protein-encoding cDNA can then beisolated by a variety of methods, such as by probing with a complete orpartial human or mouse cDNA or with one or more sets of degenerateprobes based on the disclosed sequences. A cDNA can also be cloned usingthe polymerase chain reaction, or PCR (Mullis et al U.S. Pat. No.4,683,202), using primers designed from the sequences disclosed herein.Within an additional method, the cDNA library can be used to transformor transfect host cells, and expression of the cDNA of interest can bedetected with an antibody to the protein. Similar techniques can also beapplied to the isolation of genomic clones. As used and claimed, thelanguage “an isolated polynucleotide which encodes a polypeptide, saidpolynucleotide being defined by SEQ ID NOs: 2, 4 12, 13, 19, 20, 25, 26,34 and 35” includes all allelic variants and species orthologs of thesepolypeptides.

The present invention also provides isolated protein polypeptides thatare substantially identical to the protein polypeptides of SEQ ID NO: 2and its species orthologs. By “isolated” is meant a protein orpolypeptide that is found in a condition other than its nativeenvironment, such as apart from blood and animal tissue. In a preferredform, the isolated polypeptide is substantially free of otherpolypeptides, particularly other polypeptides of animal origin. It ispreferred to provide the polypeptides in a highly purified form, i.e.greater than 95% pure, more preferably greater than 99% pure. The term“substantially identical” is used herein to denote polypeptides having50%, preferably 60%, more preferably at least 80%, sequence identity tothe sequence shown in SEQ ID NOs: 2, 4 12, 13, 19, 20, 25, 26, 34 and35, or their species orthologs. Such polypeptides will more preferablybe at least 90% identical, and most preferably 95% or more identical toSEQ ID NO:2, or its species orthologs. Percent sequence identity isdetermined by conventional methods. See, for example, Altschul et al.,Bull Math. Bio. 48: 603-616 (1986) and Henikoff and Henikoff, Proc.Natl. Acad. Sci. USA 89:10915-10919 (1992). Briefly, two amino acidsequences are aligned to optimize the alignment scores using a gapopening penalty of 10, a gap extension penalty of 1, and the “blossom62” scoring matrix of Henikoff and Henikoff (ibid.) as shown in Table 1(amino acids are indicated by the standard one-letter codes). Thepercent identity is then calculated as:

$\frac{{Total}\mspace{14mu}{number}\mspace{14mu}{of}\mspace{14mu}{identical}\mspace{14mu}{matches}}{\begin{matrix}\begin{matrix}\left\lbrack {{length}\mspace{14mu}{of}\mspace{14mu}{the}\mspace{14mu}{longer}\mspace{14mu}{sequence}\mspace{14mu}{plus}\mspace{14mu}{the}} \right. \\{{number}\mspace{14mu}{of}\mspace{14mu}{gaps}\mspace{14mu}{introduced}\mspace{14mu}{into}\mspace{14mu}{the}\mspace{14mu}{longer}}\end{matrix} \\\left. {{sequence}\mspace{14mu}{in}\mspace{14mu}{order}\mspace{14mu}{to}\mspace{14mu}{align}\mspace{14mu}{the}\mspace{14mu}{two}\mspace{14mu}{sequences}} \right\rbrack\end{matrix}} \times 100$

TABLE 1 A R N D C Q E G H I L K M F P S T W Y V A 4 R −1 5 N −2 0 6 D −2−2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 2 5 G 0 −2 0 −1 −3−2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3 −4 −3 4 L −1 −2−3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 M −1 −1 −2 −3−1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 P −1 −2−2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1−2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W −3 −3 −4 −4−2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1 −2 −3 2 −1−1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1 −2 −2 0−3 −1 4

Sequence identity of polynucleotide molecules is determined by similarmethods using a ratio as disclosed above.

Variant Zcyto10 polypeptides or substantially identical proteins andpolypeptides are characterized as having one or more amino acidsubstitutions, deletions or additions. These changes are preferably of aminor nature, that is conservative amino acid substitutions (see Table2) and other substitutions that do not significantly affect the foldingor activity of the protein or polypeptide; small deletions, typically ofone to about 30 amino acids; and small amino- or carboxyl-terminalextensions, such as an amino-terminal methionine residue, a small linkerpeptide of up to about 20-25 residues, or a small extension thatfacilitates purification (an affinity tag), such as a poly-histidinetract, protein A, Nilsson et al., EMBO J. 4:1075 (1985); Nilsson et al.,Methods Enzymol. 198:3 (1991), glutathione S transferase, Smith andJohnson, Gene 67:31 (1988), or other antigenic epitope or bindingdomain. See, in general Ford et al., Protein Expression and Purification2: 95-107 (1991). DNAs encoding affinity tags are available fromcommercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).

TABLE 2 Conservative amino acid substitutions Basic: arginine lysinehistidine Acidic: glutamic acid aspartic acid Polar: glutamineasparagine Hydrophobic: leucine isoleucine valine Aromatic:phenylalanine tryptophan tyrosine Small: glycine alanine serinethreonine methionine

The present invention further provides a variety of other polypeptidefusions [and related multimeric proteins comprising one or morepolypeptide fusions]. For example, a Zcyto10 polypeptide can be preparedas a fusion to a dimerizing protein as disclosed in U.S. Pat. Nos.5,155,027 and 5,567,584. Preferred dimerizing proteins in this regardinclude immunoglobulin constant region domains. Immunoglobulin-Zcyto10polypeptide fusions can be expressed in genetically engineered cells [toproduce a variety of multimeric Zcyto10 analogs]. Auxiliary domains canbe fused to Zcyto10 polypeptides to target them to specific cells,tissues, or macromolecules (e.g., collagen). For example, a Zcyto10polypeptide or protein could be targeted to a predetermined cell type byfusing a polypeptide to a ligand that specifically binds to a receptoron the surface of the target cell. In this way, polypeptides andproteins can be targeted for therapeutic or diagnostic purposes. AZcyto10 polypeptide can be fused to two or more moieties, such as anaffinity tag for purification and a targeting domain. Polypeptidefusions can also comprise one or more cleavage sites, particularlybetween domains. See, Tuan et al., Connective Tissue Research 34:1-9(1996).

The proteins of the present invention can also comprise non-naturallyoccurring amino acid residues. Non-naturally occurring amino acidsinclude, without limitation, trans-3-methylproline, 2,4-methanoproline,cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine,allo-threonine, methylthreonine, hydroxyethylcysteine,hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid,thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline,3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine,3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.Several methods are known in the art for incorporating non-naturallyoccurring amino acid residues into proteins. For example, an in vitrosystem can be employed wherein nonsense mutations are suppressed usingchemically aminoacylated suppressor tRNAs. Methods for synthesizingamino acids and aminoacylating tRNA are known in the art. Essentialamino acids in the polypeptides of the present invention can beidentified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis [Cunninghamand Wells, Science 244: 1081-1085 (1989)]; Bass et al., Proc. Natl.Acad. Sci. USA 88:4498-4502 (1991). In the latter technique, singlealanine mutations are introduced at every residue in the molecule, andthe resultant mutant molecules are tested for biological activity (e.g.,ligand binding and signal transduction) to identify amino acid residuesthat are critical to the activity of the molecule. Sites ofligand-protein interaction can also be determined by analysis of crystalstructure as determined by such techniques as nuclear magneticresonance, crystallography or photoaffinity labeling. See, for example,de Vos et al., Science 255:306-312 (1992); Smith et al., J. Mol. Biol.224:899-904 (1992); Wlodaver et al., FEBS Lett. 309:59-64 (1992). Theidentities of essential amino acids can also be inferred from analysisof homologies with related proteins.

Multiple amino acid substitutions can be made and tested using knownmethods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer, Science 241:53-57 (1988) or Bowie and SauerProc. Natl. Acad. Sci. USA 86:2152-2156 (1989). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832-10837 (1991);Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO92/06204) and region-directed mutagenesis, Derbyshire et al., Gene46:145 (1986); Ner et al., DNA 7:127 (1988).

Mutagenesis methods as disclosed above can be combined withhigh-throughput screening methods to detect activity of cloned,mutagenized proteins in host cells. Preferred assays in this regardinclude cell proliferation assays and biosensor-based ligand-bindingassays, which are described below. Mutagenized DNA molecules that encodeactive proteins or portions thereof (e.g., ligand-binding fragments) canbe recovered from the host cells and rapidly sequenced using modernequipment. These methods allow the rapid determination of the importanceof individual amino acid residues in a polypeptide of interest, and canbe applied to polypeptides of unknown structure.

Using the methods discussed above, one of ordinary skill in the art canprepare a variety of polypeptides that are substantially identical toSEQ ID NOs: 2, 4 12, 13, 19, 20, 25, 26, 34 and 35 or allelic variantsthereof and retain the properties of the wild-type protein. As expressedand claimed herein the language, “a polypeptide as defined by SEQ ID NO:2” includes all allelic variants and species orthologs of thepolypeptide.

The protein polypeptides of the present invention, including full-lengthproteins, protein fragments (e.g. ligand-binding fragments), and fusionpolypeptides can be produced in genetically engineered host cellsaccording to conventional techniques. Suitable host cells are those celltypes that can be transformed or transfected with exogenous DNA andgrown in culture, and include bacteria, fungal cells, and culturedhigher eukaryotic cells. Eukaryotic cells, particularly cultured cellsof multicellular organisms, are preferred. Techniques for manipulatingcloned DNA molecules and introducing exogenous DNA into a variety ofhost cells are disclosed by Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989), and Ausubel et al., ibid.

Polynucleotides, generally a cDNA sequence, of the present inventionencode the above-described polypeptides. A DNA sequence which encodes apolypeptide of the present invention is comprised of a series of codons,each amino acid residue of the polypeptide being encoded by a codon andeach codon being comprised of three nucleotides. The amino acid residuesare encoded by their respective codons as follows.

-   -   Alanine (Ala) is encoded by GCA, GCC, GCG or GCT;    -   Cysteine (Cys) is encoded by TGC or TGT;    -   Aspartic acid (Asp) is encoded by GAC or GAT;    -   Glutamic acid (Glu) is encoded by GAA or GAG;    -   Phenylalanine (Phe) is encoded by TTC or TTT;    -   Glycine (Gly) is encoded by GGA, GGC, GGG or GGT;    -   Histidine (His) is encoded by CAC or CAT;    -   Isoleucine (Ile) is encoded by ATA, ATC or ATT;    -   Lysine (Lys) is encoded by AAA, or AAG;    -   Leucine (Leu) is encoded by TTA, TTG, CTA, CTC, CTG or CTT;    -   Methionine (Met) is encoded by ATG;    -   Asparagine (Asn) is encoded by AAC or AAT;    -   Proline (Pro) is encoded by CCA, CCC, CCG or CCT;    -   Glutamine (Gln) is encoded by CAA or CAG;    -   Arginine (Arg) is encoded by AGA, AGG, CGA, CGC, CGG or CGT;    -   Serine (Ser) is encoded by AGC, AGT, TCA, TCC, TCG or TCT;    -   Threonine (Thr) is encoded by ACA, ACC, ACG or ACT;    -   Valine (Val) is encoded by GTA, GTC, GTG or GTT;    -   Tryptophan (Trp) is encoded by TGG; and    -   Tyrosine (Tyr) is encoded by TAC or TAT.

It is to be recognized that according to the present invention, when acDNA is claimed as described above, it is understood that what isclaimed are both the sense strand, the anti-sense strand, and the DNA asdouble-stranded having both the sense and anti-sense strand annealedtogether by their respective hydrogen bonds. Also claimed is themessenger RNA (mRNA) which encodes the polypeptides of the presentinvention, and which mRNA is encoded by the above-described cDNA. Amessenger RNA (mRNA) will encode a polypeptide using the same codons asthose defined above, with the exception that each thymine nucleotide (T)is replaced by a uracil nucleotide (U).

In general, a DNA sequence encoding a Zcyto10 polypeptide is operablylinked to other genetic elements required for its expression, generallyincluding a transcription promoter and terminator, within an expressionvector. The vector will also commonly contain one or more selectablemarkers and one or more origins of replication, although those skilledin the art will recognize that within certain systems selectable markersmay be provided on separate vectors, and replication of the exogenousDNA may be provided by integration into the host cell genome. Selectionof promoters, terminators, selectable markers, vectors and otherelements is a matter of routine design within the level of ordinaryskill in the art. Many such elements are described in the literature andare available through commercial suppliers.

To direct a Zcyto10 polypeptide into the secretory pathway of a hostcell, a secretory signal sequence (also known as a leader sequence,prepro sequence or pre sequence) is provided in the expression vector.The secretory signal sequence may be that of the protein, or may bederived from another secreted protein (e.g., t-PA) or synthesized denovo. The secretory signal sequence is joined to the Zcyto10 DNAsequence in the correct reading frame. Secretory signal sequences arecommonly positioned 5′ to the DNA sequence encoding the polypeptide ofinterest, although certain signal sequences may be positioned elsewherein the DNA sequence of interest (see, e.g., Welch et al., U.S. Pat. No.5,037,743; Holland et al., U.S. Pat. No. 5,143,830).

Methods for introducing exogenous DNA into mammalian host cells includecalcium phosphate-mediated transfection, Wigler et al., Cell 14:725(1978); Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981: Grahamand Van der Eb, Virology 52:456 (1973), electroporation, Neumann et al.,EMBO J. 1:841-845 (1982), DEAE-dextran mediated transfection, Ausubel etal., eds., Current Protocols in Molecular Biology, (John Wiley and Sons,Inc., NY, 1987), and liposome-mediated transfection, Hawley-Nelson etal., Focus 15:73 (1993); Ciccarone et al., Focus 15:80 (1993). Theproduction of recombinant polypeptides in cultured mammalian cells isdisclosed, for example, by Levinson et al., U.S. Pat. No. 4,713,339;Hagen et al., U.S. Pat. No. 4,784,950; Palmiter et al., U.S. Pat. No.4,579,821; and Ringold, U.S. Pat. No. 4,656,134. Suitable culturedmammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No.CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293[ATCC No. CRL 1573; Graham et al., J. Gen. Virol. 36:59-72 (1977) andChinese hamster ovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines.Additional suitable cell lines are known in the art and available frompublic depositories such as the American Type Culture Collection,Rockville, Md. In general, strong transcription promoters are preferred,such as promoters from SV-40 or cytomegalovirus. See, e.g., U.S. Pat.No. 4,956,288. Other suitable promoters include those frommetallothionein genes (U.S. Pat. Nos. 4,579,821 and 4,601,978 and theadenovirus major late promoter.

Drug selection is generally used to select for cultured mammalian cellsinto which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants”. Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.” Apreferred selectable marker is a gene encoding resistance to theantibiotic neomycin. Selection is carried out in the presence of aneomycin-type drug, such as G-418 or the like. Selection systems mayalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.A preferred amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g. hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used. Alternative markers that introducean altered phenotype, such as green fluorescent protein, or cell surfaceproteins such as CD4, CD8, Class I MHC, placental alkaline phosphatasemay be used to sort transfected cells from untransfected cells by suchmeans as FACS sorting or magnetic bead separation technology.

Other higher eukaryotic cells can also be used as hosts, includinginsect cells, plant cells and avian cells. Transformation of insectcells and production of foreign polypeptides therein is disclosed byGuarino et al., U.S. Pat. No. 5,162,222; Bang et al., U.S. Pat. No.4,775,624; and WIPO publication WO 94/06463. The use of Agrobacteriumrhizogenes as a vector for expressing genes in plant cells has beenreviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47-58 (1987).Insect cells can be infected with recombinant baculovirus, commonlyderived from Autographa californica nuclear polyhedrosis virus (AcNPV).See, King, L. A. and Possee, R. D., The Baculovirus Expression System: ALaboratory Guide (Chapman & Hall, London); O'Reilly, D. R. et al.,Baculovirus Expression Vectors: A Laboratory Manual (University Press,New York, Oxford, 1994); and, Richardson, C. D., Ed., BaculovirusExpression Protocols. Methods in Molecular Biology, (Humana Press,Totowa, N.J., 1995). A second method of making recombinant Zcyto10baculovirus utilizes a transposon-based system described by Luckow, V.A, et al., J Virol 67:4566-79 1993). This system, which utilizestransfer vectors, is sold in the Bac-to-Bac™ kit (Life Technologies,Rockville, Md.). This system utilizes a transfer vector, pFastBac1™(Life Technologies) containing a Tn7 transposon to move the DNA encodingthe Zcyto10 polypeptide into a baculovirus genome maintained in E. colias a large plasmid called a “bacmid.” See, Hill-Perkins, M. S, andPossee, R. D., J Gen Virol 71:971-6, (1990); Bonning, B. C. et al., JGen Virol 75:1551-6 (1994); and, Chazenbalk, G. D., and Rapoport, B., JBiol Chem 270:1543-9 (1995). In addition, transfer vectors can includean in-frame fusion with DNA encoding an epitope tag at the C- orN-terminus of the expressed Zcyto10 polypeptide, for example, a Glu-Gluepitope tag, Grussenmeyer, T. et al., Proc. Natl. Acad. Sci. 82:7952-4(1985). Using a technique known in the art, a transfer vector containingZcyto10 is transformed into E. Coli, and screened for bacmids whichcontain an interrupted lacZ gene indicative of recombinant baculovirus.The bacmid DNA containing the recombinant baculovirus genome isisolated, using common techniques, and used to transfect Spodopterafrugiperda cells, e.g. Sf9 cells. Recombinant virus that expressesZcyto10 is subsequently produced. Recombinant viral stocks are made bymethods commonly used the art.

The recombinant virus is used to infect host cells, typically a cellline derived from the fall armyworm, Spodoptera frugiperda. See, ingeneral, Glick and Pasternak, Molecular Biotechnology: Principles andApplications of Recombinant DNA, ASM Press, Washington, D.C. (1994).Another suitable cell line is the High FiveO™ cell line (Invitrogen)derived from Trichoplusia ni (U.S. Pat. No. 5,300,435). Commerciallyavailable serum-free media are used to grow and maintain the cells.Suitable media are Sf900II™ (Life Technologies) or ESF 921™ (ExpressionSystems) for the Sf9 cells; and Ex-cellO405™ (JRH Biosciences, Lenexa,Kans.) or Express FiveO™ (Life Technologies) for the T. ni cells. Thecells are grown up from an inoculation density of approximately 2-5×10⁵cells to a density of 1-2×10⁶ cells at which time a recombinant viralstock is added at a multiplicity of infection (MOI) of 0.1 to 10, moretypically near 3. Procedures used are generally described in availablelaboratory manuals (King, L. A. and Possee, R. D., ibid.; O'Reilly, D.R. et al., ibid.; Richardson, C. D., ibid.). Subsequent purification ofthe Zcyto10 polypeptide from the supernatant can be achieved usingmethods described herein.

Fungal cells, including yeast cells, and particularly cells of the genusSaccharomyces, can also be used within the present invention, such asfor producing protein fragments or polypeptide fusions. Methods fortransforming yeast cells with exogenous DNA and producing recombinantpolypeptides therefrom are disclosed by, for example, Kawasaki, U.S.Pat. No. 4,599,311; Kawasaki et al., U.S. Pat. No. 4,931,373; Brake,U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat. No. 5,037,743; andMurray et al., U.S. Pat. No. 4,845,075. Transformed cells are selectedby phenotype determined by the selectable marker, commonly drugresistance or the ability to grow in the absence of a particularnutrient (e.g., leucine). A preferred vector system for use in yeast isthe POT1 vector system disclosed by Kawasaki et al. (U.S. Pat. No.4,931,373), which allows transformed cells to be selected by growth inglucose-containing media. Suitable promoters and terminators for use inyeast include those from glycolytic enzyme genes (see, e.g., Kawasaki,U.S. Pat. No. 4,599,311; Kingsman et al., U.S. Pat. No. 4,615,974; andBitter, U.S. Pat. No. 4,977,092 and alcohol dehydrogenase genes. Seealso U.S. Pat. Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454.Transformation systems for other yeasts, including Hansenula polymorpha,Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis,Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichiaguillermondii and Candida maltosa are known in the art. See, forexample, Gleeson et al., J. Gen. Microbiol. 132:3459-3465 (1986) andCregg, U.S. Pat. No. 4,882,279. Aspergillus cells may be utilizedaccording to the methods of McKnight et al., U.S. Pat. No. 4,935,349.Methods for transforming Acremonium chrysogenum are disclosed by Suminoet al., U.S. Pat. No. 5,162,228. Methods for transforming Neurospora aredisclosed by Lambowitz, U.S. Pat. No. 4,486,533.

The use of Pichia methanolica as host for the production of recombinantproteins is disclosed in WIPO Publications WO 97/17450, WO 97/17451, WO98/02536, and WO 98/02565. DNA molecules for use in transforming P.methanolica will commonly be prepared as double-stranded, circularplasmids, which are preferably linearized prior to transformation. Forpolypeptide production in P. methanolica, it is preferred that thepromoter and terminator in the plasmid be that of a P. methanolica gene,such as a P. methanolica alcohol utilization gene (AUG1 or AUG2). Otheruseful promoters include those of the dihydroxyacetone synthase (DHAS),formate dehydrogenase (FMD), and catalase (CAT) genes. To facilitateintegration of the DNA into the host chromosome, it is preferred to havethe entire expression segment of the plasmid flanked at both ends byhost DNA sequences. A preferred selectable marker for use in Pichiamethanolica is a P. methanolica ADE2 gene, which encodesphosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), whichallows ade2 host cells to grow in the absence of adenine. Forlarge-scale, industrial processes where it is desirable to minimize theuse of methanol, it is preferred to use host cells in which bothmethanol utilization genes (AUG1 and AUG2) are deleted. For productionof secreted proteins, host cells deficient in vacuolar protease genes(PEP4 and PRB1) are preferred. Electroporation is used to facilitate theintroduction of a plasmid containing DNA encoding a polypeptide ofinterest into P. methanolica cells. It is preferred to transform P.methanolica cells by electroporation using an exponentially decaying,pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm,preferably about 3.75 kV/cm, and a time constant (t) of from 1 to 40milliseconds, most preferably about 20 milliseconds.

Prokaryotic host cells, including strains of the bacteria Escherichiacoli, Bacillus and other genera are also useful host cells within thepresent invention. Techniques for transforming these hosts andexpressing foreign DNA sequences cloned therein are well known in theart (see, e.g., Sambrook et al., ibid.). When expressing a Zcyto10polypeptide in bacteria such as E. coli, the polypeptide may be retainedin the cytoplasm, typically as insoluble granules, or may be directed tothe periplasmic space by a bacterial secretion sequence. In the formercase, the cells are lysed, and the granules are recovered and denaturedusing, for example, guanidine isothiocyanate or urea. The denaturedpolypeptide can then be refolded and dimerized by diluting thedenaturant, such as by dialysis against a solution of urea and acombination of reduced and oxidized glutathione, followed by dialysisagainst a buffered saline solution. In the latter case, the polypeptidecan be recovered from the periplasmic space in a soluble and functionalform by disrupting the cells (by, for example, sonication or osmoticshock) to release the contents of the periplasmic space and recoveringthe protein, thereby obviating the need for denaturation and refolding.

Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell. P. methanolicacells are cultured in a medium comprising adequate sources of carbon,nitrogen and trace nutrients at a temperature of about 25° C. to 35° C.Liquid cultures are provided with sufficient aeration by conventionalmeans, such as shaking of small flasks or sparging of fermentors. Apreferred culture medium for P. methanolica is YEPD (2% D-glucose, 2%Bacto™ Peptone (Difco Laboratories, Detroit, Mich.), 1% Bacto™ yeastextract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine).

Within one aspect of the present invention, a novel protein is producedby a cultured cell, and the cell is used to screen for a receptor orreceptors for the protein, including the natural receptor, as well asagonists and antagonists of the natural ligand.

Protein Isolation:

It is preferred to purify the polypeptides of the present invention to≧80% purity, more preferably to ≧90% purity, even more preferably ≧95%purity, and particularly preferred is a pharmaceutically pure state,that is greater than 99.9% pure with respect to contaminatingmacromolecules, particularly other proteins and nucleic acids, and freeof infectious and pyrogenic agents. Preferably, a purified polypeptideis substantially free of other polypeptides, particularly otherpolypeptides of animal origin.

Expressed recombinant polypeptides (or chimeric polypeptides) can bepurified using fractionation and/or conventional purification methodsand media. Ammonium sulfate precipitation and acid or chaotropeextraction may be used for fractionation of samples. Exemplarypurification steps may include hydroxyapatite, size exclusion, FPLC andreverse-phase high performance liquid chromatography. Suitable anionexchange media include derivatized dextrans, agarose, cellulose,polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Qderivatives are preferred, with DEAE Fast-Flow Sepharose (Pharmacia,Piscataway, N.J.) being particularly preferred. Exemplarychromatographic media include those media derivatized with phenyl,butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia),Toyopearl butyl 650 (Toso Haas, Montgomeryville, Pa.), Octyl-Sepharose(Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG71 (Toso Haas) and the like. Suitable solid supports include glassbeads, silica-based resins, cellulosic resins, agarose beads,cross-linked agarose beads, polystyrene beads, cross-linkedpolyacrylamide resins and the like that are insoluble under theconditions in which they are to be used. These supports may be modifiedwith reactive groups that allow attachment of proteins by amino groups,carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydratemoieties. Examples of coupling chemistries include cyanogen bromideactivation, N-hydroxysuccinimide activation, epoxide activation,sulfhydryl activation, hydrazide activation, and carboxyl and aminoderivatives for carbodiimide coupling chemistries. These and other solidmedia are well known and widely used in the art, and are available fromcommercial suppliers. Methods for binding receptor polypeptides tosupport media are well known in the art. Selection of a particularmethod is a matter of routine design and is determined in part by theproperties of the chosen support. See, for example, AffinityChromatography: Principles & Methods (Pharmacia LKB Biotechnology,Uppsala, Sweden, 1988).

The polypeptides of the present invention can be isolated byexploitation of their properties. For example, immobilized metal ionadsorption (IMAC) chromatography can be used to purify histidine-richproteins. Briefly, a gel is first charged with divalent metal ions toform a chelate (E. Sulkowski, Trends in Biochem. 3:1-7 (1985).Histidine-rich proteins will be adsorbed to this matrix with differingaffinities, depending upon the metal ion used, and will be eluted bycompetitive elution, lowering the pH, or use of strong chelating agents.Other methods of purification include purification of glycosylatedproteins by lectin affinity chromatography and ion exchangechromatography (Methods in Enzymol., Vol. 182, “Guide to ProteinPurification”, M. Deutscher, (ed.), pp. 529-539 (Acad. Press, San Diego,1990. Alternatively, a fusion of the polypeptide of interest and anaffinity tag (e.g., polyhistidine, maltose-binding protein, animmunoglobulin domain) may be constructed to facilitate purification.

Uses

The polypeptide of the present invention has the structuralcharacteristics of a four-helix bundle cytokine. A protein is generallycharacterized as a cytokine by virtue of its solubility and ability toact via cell surface receptors to signal and modulate cellproliferation. Cytokines fall into several tertiary structural foldclasses, including cysteine-rich dimers (e.g., insulin, PDGF),beta-trefoil folds (e.g., FGF, IL-1), and all-alpha four helix bundles.The latter are characterized by four helices, labeled A, B, C and D, ina unique up-up-down-down topology, where two overhand loops link helicesA and B and helices C and D. See, for example, Manavalan et al., Journalof protein Chemistry 11(3): 321-31, (1992). The four-helix bundlecytokines are sometimes further subdivided into short chain (e.g., IL-4,Il-2, GM-CSF) and long chain (e.g., TPO, growth hormone, leptin, IL-10),where the latter generally display longer A and D helices and overhandloops. Henceforth we shall use the term “cytokine” synonymously with“four-helix bundle cytokine”. Helix A of zcyto10 includes amino acidresidue 35, an isoleucine, through amino acid residue 49, an isoleucine,also defined by SEQ ID NO:14; helix B includes amino acid 91, a leucine,through amino acid 105, a threonine, also defined by SEQ ID NO:15; helixC includes amino acid residue 112, a leucine, through amino acid residue126, a cysteine, also defined by SEQ ID NO:16; helix D includes aminoacid residue 158, a valine, through amino acid residue 172, amethionine, also defined by SEQ ID NO:17.

Human Zcyto10 has an intramolecular disulfide bond between Cys33 andCys126. The other four cysteines, Cys80, Cys132, Cys81 and Cys134 arepredicted to form two intramolecular disulfide bonds in the arrangementCys80-Cys 132 and Cys81-Cys 134. Residues that are predicted to becrucial for the structural stability of Zcyto10 include Cys33, Cys126,Cys80, Cys132, Cys81 and Cys 134. Mutation of any one of these residuesto any other residue is expected to inactivate the function of Zcyto10.

The structural stability of Zcyto10 is also dependent on the maintenanceof a buried hydrophobic face on the four alpha helices. Residues Ile42,Phe46, Ile49, Leu91, Val94, Phe95, Tyr98, Leu112, Phe116, Ile119,Leu123, Val158, Leu162, Leu165, Leu168, Leu169 and Met172 are predictedto be buried in the core of the protein and if they are changed, thesubstituted amino acid residue must be a hydrophobic amino acid.

Residues expected to be involved in binding of Zcyto10 to a cell surfacereceptor include Asp57, on the overhand loop between helix A and B, andLys160 and Glu164, charged residues predicted to be exposed on thesurface of helix D. On the surface of the protein, on the loop AB andhelix D areas, is a hydrophobic surface patch comprising residues Ile62,Leu71, Ile167, and Trp 171. These residues may interact with ahydrophobic surface patch on a cell surface receptor.

The human Zcyto10 polypeptide of the present invention has about a 28%identity to interleukin-10 (IL-10). Mouse Zcyto10 polypeptide hasapproximately 24% identity to human IL-10, and about 27% identity tomouse IL-10. Human Zcyto10 polypeptide has approximately 76% identitywith mouse Zcyto10 polypeptide.

Helix A of mouse Zcyto10 includes amino acid residue 35, an isoleucine,through amino acid residue 49, an arginine, of SEQ ID NO: 19, alsodefined by SEQ ID NO:21. Helix B of mouse Zcyto10 includes amino acidresidue 91, a leucine, through amino acid residue 105, a threonine, ofSEQ ID NO: 19, also defined by SEQ ID NO:22. Helix C of mouse Zcyto10includes amino acid residue 112, a leucine, through amino acid residue126, a cysteine, of SEQ ID NO: 19, also defined by SEQ ID NO:23. Helix Dof mouse Zcyto10 includes amino acid residue 158, a valine, throughamino acid residue 172, a methionine, of SEQ ID NO: 19, also defined bySEQ ID NO:24.

IL-10 is a cytokine that inhibits production of other cytokines, inducesproliferation and differentiation of activated B lymphocytes, inhibitsHIV-1 replication and exhibits antagonistic effects on gamma interferon.IL-10 appears to exist as a dimer formed from two alpha-helicalpolypeptide regions related by a 180° rotation. See, for example, Zdanovet al., Structure: 3(6): 591-601 (1996). IL-10 has been reported to be aproduct of activated Th2 T-cells, B-cells, keratinocytes andmonocytes/macrophages that is capable of modulating a Th1 T-cellresponse. Such modulation may be accomplished by inhibiting cytokinesynthesis by Th1 T-cells. See, for example, Hus et al., Int. Immunol. 4:563 (1992) and D'Andrea et al., J. Exp. Med. 178: 1042 (1992). IL-10 hasalso been reported to inhibit cytokine synthesis by natural killer cellsand monocytes/macrophages. See, for example, Hus et al. cited above andFiorentino et al., J. Immunol. 146: 3444 (1991). In addition, IL-10 hasbeen found to have a protective effect with respect to insulin dependentdiabetes mellitus.

In analysis of the tissue distribution of the mRNA corresponding to thisnovel DNA, a single transcript was observed at approximately 1.2 kb.Using Clontech Multiple Tissue Northerns, the human transcript wasapparent in trachea, placenta, testis, skin, salivary gland, prostate,thyroid with less expression observed in stomach and pancreas. Zcyto10was expressed in the following mouse tissues: kidney, skeletal muscle,salivary gland, liver and skin.

The tissue specificity of Zcyto10 expression suggests that Zcyto10 maybe a growth and/or maintenance factor in the trachea and salivaryglands, stomach, pancreas and muscle; and may be important in localimmune responses. Also, the Zcyto10 gene's location on chromosome 1q32.2indicates that Zcyto10 is a growth/differentiation factor or importantin regulating the immune response as IL-10.

The present invention also provides reagents which will find use indiagnostic applications. A probe comprising the Zcyto10 DNA or RNA or asubsequence thereof can be used to determine if the Zcyto10 gene ispresent on chromosome 1 or if a mutation has occurred.

The present invention also provides reagents with significanttherapeutic value. The Zcyto10 polypeptide (naturally occurring orrecombinant), fragments thereof, antibodies and anti-idiotypicantibodies thereto, along with compounds identified as having bindingaffinity to the Zcyto10 polypeptide, should be useful in the treatmentof conditions associated with abnormal physiology or development,including abnormal proliferation, e.g., cancerous conditions, ordegenerative conditions or altered immunity.

Antibodies to the Zcyto10 polypeptide can be purified and thenadministered to a patient. These reagents can be combined fortherapeutic use with additional active or inert ingredients, e.g., inpharmaceutically acceptable carriers or diluents along withphysiologically innocuous stabilizers and excipients. These combinationscan be sterile filtered and placed into dosage forms as bylyophilization in dosage vials or storage in stabilized aqueouspreparations. This invention also contemplates use of antibodies,binding fragments thereof or single-chain antibodies of the antibodiesincluding forms which are not complement binding.

The quantities of reagents necessary for effective therapy will dependupon many different factors, including means of administration, targetsite, physiological state of the patient, and other medicationsadministered. Thus, treatment dosages should be titrated to optimizesafety and efficacy. Typically, dosages used in vitro may provide usefulguidance in the amounts useful for in vivo administration of thesereagents. Animal testing of effective doses for treatment of particulardisorders will provide further predictive indication of human dosage.Methods for administration include oral, intravenous, peritoneal,intramuscular, transdermal or administration into the lung or trachea inspray form by means or a nebulizer or atomizer. Pharmaceuticallyacceptable carriers will include water, saline, buffers to name just afew. Dosage ranges would ordinarily be expected from 1 μg to 1000 μg perkilogram of body weight per day. However, the doses by be higher orlower as can be determined by a medical doctor with ordinary skill inthe art. For a complete discussion of drug formulations and dosageranges see Remington's Pharmaceutical Sciences, 18^(th) Ed., (MackPublishing Co., Easton, Pa., 1996), and Goodman and Gilman's: ThePharmacological Bases of Therapeutics, 9^(th) Ed. (Pergamon Press 1996).

Nucleic Acid-based Therapeutic Treatment

If a mammal has a mutated or lacks a Zcyto10 gene, the Zcyto10 gene canbe introduced into the cells of the mammal. In one embodiment, a geneencoding a Zcyto10 polypeptide is introduced in vivo in a viral vector.Such vectors include an attenuated or defective DNA virus, such as butnot limited to herpes simplex virus (HSV), papillomavirus, Epstein Barrvirus (EBV), adenovirus, adeno-associated virus (AAV), and the like.Defective viruses, which entirely or almost entirely lack viral genes,are preferred. A defective virus is not infective after introductioninto a cell. Use of defective viral vectors allows for administration tocells in a specific, localized area, without concern that the vector caninfect other cells. Examples of particular vectors include, but are notlimited to, a defective herpes virus 1 (HSV1) vector [Kaplitt et al.,Molec. Cell. Neurosci., 2:320-330 (1991)], an attenuated adenovirusvector, such as the vector described by Stratford-Perricaudet et al., J.Clin. Invest., 90:626-630 (1992), and a defective adeno-associated virusvector [Samulski et al., J. Virol., 61:3096-3101 (1987); Samulski et al.J. Virol, 63:3822-3828 (1989)].

In another embodiment, the gene can be introduced in a retroviralvector, e.g., as described in Anderson et al., U.S. Pat. No. 5,399,346;Mann et al., Cell, 33:153 (1983); Temin et al., U.S. Pat. No. 4,650,764;Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., J. Virol.,62:1120 (1988); Temin et al., U.S. Pat. No. 5,124,263; InternationalPatent Publication No. WO 95/07358, published Mar. 16, 1995 by Doughertyet al.; and Blood, 82:845 (1993). Alternatively, the vector can beintroduced by lipofection in vivo using liposomes. Synthetic cationiclipids can be used to prepare liposomes for in vivo transfection of agene encoding a marker [Felgner et al., Proc. Natl. Acad. Sci. USA,84:7413-7417 (1987); see Mackey et al., Proc. Natl. Acad. Sci. USA,85:8027-8031 (1988)]. The use of lipofection to introduce exogenousgenes into specific organs in vivo has certain practical advantages.Molecular targeting of liposomes to specific cells represents one areaof benefit. It is clear that directing transfection to particular cellsrepresents one area of benefit. It is clear that directing transfectionto particular cell types would be particularly advantageous in a tissuewith cellular heterogeneity, such as the pancreas, liver, kidney, andbrain. Lipids may be chemically coupled to other molecules for thepurpose of targeting. Targeted peptides, e.g., hormones orneurotransmitters, and proteins such as antibodies, or non-peptidemolecules could be coupled to liposomes chemically. These liposomes canalso be administered in spray form into the lung or trachea by means ofan atomizer or nebulizer.

It is possible to remove the cells from the body and introduce thevector as a naked DNA plasmid and then re-implant the transformed cellsinto the body. Naked DNA vector for gene therapy can be introduced intothe desired host cells by methods known in the art, e.g., transfection,electroporation, microinjection, transduction, cell fusion, DEAEdextran, calcium phosphate precipitation, use of a gene gun or use of aDNA vector transporter [see, e.g., Wu et al., J. Biol. Chem.,267:963-967 (1992); Wu et al., J. Biol. Chem., 263:14621-14624 (1988)].

Zcyto10 polypeptides can also be used to prepare antibodies thatspecifically bind to Zcyto10 polypeptides. These antibodies can then beused to manufacture anti-idiotypic antibodies. As used herein, the term“antibodies” includes polyclonal antibodies, monoclonal antibodies,antigen-binding fragments thereof such as F(ab′)₂ and Fab fragments, andthe like, including genetically engineered antibodies. Antibodies aredefined to be specifically binding if they bind to a Zcyto10 polypeptidewith a K_(a) of greater than or equal to 10⁷/M. The affinity of amonoclonal antibody can be readily determined by one of ordinary skillin the art (see, for example, Scatchard, ibid.).

Methods for preparing polyclonal and monoclonal antibodies are wellknown in the art (see for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition (Cold Spring Harbor, N.Y., 1989); andHurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques andApplications (CRC Press, Inc., Boca Raton, Fla., 1982), which areincorporated herein by reference). As would be evident to one ofordinary skill in the art, polyclonal antibodies can be generated from avariety of warm-blooded animals such as horses, cows, goats, sheep,dogs, chickens, rabbits, mice, and rats. The immunogenicity of a Zcyto10polypeptide may be increased through the use of an adjuvant such asFreund's complete or incomplete adjuvant. A variety of assays known tothose skilled in the art can be utilized to detect antibodies whichspecifically bind to Zcyto10 polypeptides. Exemplary assays aredescribed in detail in Antibodies: A Laboratory Manual, Harlow and Lane(Eds.), (Cold Spring Harbor Laboratory Press, 1988). Representativeexamples of such assays include: concurrent immunoelectrophoresis,radio-immunoassays, radio-immunoprecipitations, enzyme-linkedimmunosorbent assays (ELISA), dot blot assays, inhibition or competitionassays, and sandwich assays.

Antibodies to Zcyto10 are may be used for tagging cells that express theprotein, for affinity purification, within diagnostic assays fordetermining circulating levels of soluble protein polypeptides, and asantagonists to block ligand binding and signal transduction in vitro andin vivo.

Within another aspect of the present invention there is provided apharmaceutical composition comprising purified Zcyto10 polypeptide incombination with a pharmaceutically acceptable vehicle. Suchcompositions may be useful for modulating of cell proliferation, celldifferentiation or cytokine production in the prevention or treatment ofconditions characterized by improper cell proliferation, celldifferentiation or cytokine production, as are further discussed herein.Moreover, Zcyto10 polypeptides of the present invention may be used intrachea-specific or tracheobronchial-specific applications, such as inthe maintenance or wound repair of the tracheobronchial epithelium orcells underlying the same, in regulating mucous production or mucocilaryclearance of debris or in treatment of asthma, bronchitis or otherdiseases of the tracheobronchial tract. It is expected that Zcyto10polypeptide would be administered at a dose ranging between the samedoses used for Zcyto10-Fc construct to doses 100-fold higher, dependingupon the stability of Zcyto10 polypeptide. Therapeutic doses of Zcyto10would range from 5 to 5000 μg/kg/day.

The Zcyto10 polypeptide of the present invention is expressed highly insalivary gland and trachea and has been found in saliva by Western blotanalysis. The salivary glands synthesize and secrete a number ofproteins having diverse biological functions. Such proteins facilitatelubrication of the oral cavity (e.g., mucins and proline-rich proteins),remineralization (e.g., statherin and ionic proline-rich proteins) anddigestion (e.g., amylase, lipase and proteases) and provideanti-microbial (e.g., proline-rich proteins, lysozyme, histatins andlactoperoxidase) and mucosal integrity maintenance (e.g., mucins)capabilities. In addition, saliva is a rich source of growth factorssynthesized by the salivary glands. For example, saliva is known tocontain epidermal growth factor (EGF), nerve growth factor (NGF),transforming growth factor-alpha (TGF-α), transforming growthfactor-beta (TGF-β), insulin, insulin-like growth factors I and II(IGF-I and IGF-II) and fibroblast growth factor (FGF). See, for example,Zelles et al., J. Dental. Res. 74(12): 1826-32, 1995. Synthesis ofgrowth factors by the salivary gland is believed to beandrogen-dependent and to be necessary for the health of the oral cavityand gastrointestinal tract.

Thus, Zcyto10 polypeptides, agonists or antagonists thereof may betherapeutically useful in the regeneration of the gastrointestinal tractor oral cavity. To verify this presence of this capability in Zcyto10polypeptides, agonists or antagonists of the present invention, suchZcyto10 polypeptides, agonists or antagonists are evaluated with respectto their ability to break down starch according to procedures known inthe art. Zcyto10 polypeptides, agonists or antagonists thereof may beuseful in the treatment of asthma and other diseases of thetracheobronchial tract, such as bronchitis and the like, by interventionin the cross-regulation of Th1 and Th2 lymphocytes, regulation ofgrowth, differentiation and cytokine production of other inflammatorycellular mediators, such as eosinophils, mast cells, basophils,neutrophils and macrophages. Zcyto10 polypeptides, agonists orantagonists thereof may also modulate muscle tone in thetracheobronchial tract.

Zcyto10 polypeptides can also be used to treat a number of skinconditions either systemically or locally when placed in an ointment orcream, for example eczema, psoriasis or dry skin conditions in generalor as related skin attentions. Also the Zcyto10 polypeptide can bedirectly injected into muscle to treat muscle atrophy in the elderly,the sick or the bed-ridden.

Radiation hybrid mapping is a somatic cell genetic technique developedfor constructing high-resolution, contiguous maps of mammalianchromosomes [Cox et al., Science 250:245-250 (1990)]. Partial or fullknowledge of a gene's sequence allows the designing of PCR primerssuitable for use with chromosomal radiation hybrid mapping panels.Commercially available radiation hybrid mapping panels which cover theentire human genome, such as the Stanford G3 RH Panel and the GeneBridge4 RH Panel (Research Genetics, Inc., Huntsville, Ala.), are available.These panels enable rapid, PCR based, chromosomal localizations andordering of genes, sequence-tagged sites (STSs), and othernonpolymorphic- and polymorphic markers within a region of interest.This includes establishing directly proportional physical distancesbetween newly discovered genes of interest and previously mappedmarkers. The precise knowledge of a gene's position can be useful in anumber of ways including: 1) determining if a sequence is part of anexisting contig and obtaining additional surrounding genetic sequencesin various forms such as YAC-, BAC- or cDNA clones, 2) providing apossible candidate gene for an inheritable disease which shows linkageto the same chromosomal region, and 3) for cross-referencing modelorganisms such as mouse which may be beneficial in helping to determinewhat function a particular gene might have.

The results showed that the Zcyto10 gene maps 889.26 cR_(—)3000 from thetop of the human chromosome 1 linkage group on the WICGR radiationhybrid map. Proximal and distal framework markers were D1S504 andWI-9641 (D1S2427), respectively. The use of the surrounding markerspositions the Zcyto10 gene in the 1q32.2 region on the integrated LDBchromosome 1 map (The Genetic Location Database, University ofSouthhampton, WWW server:http://cedar.genetics.soton.ac.uk/public_html/). Numerous genes havebeen mapped to the 1q32.2 region of chromosome 1. In particular,mutations in this region have been found to result in van der Woudesyndrome, associated with malformation of the lower lip that issometimes associated with cleft palate. Thus, the Zcyto10 gene, which isexpressed in the salivary gland, may be used in gene therapy of thissyndrome. If a mammal has a mutated or lacks a Zcyto10 gene, the Zcyto10gene can be introduced into the cells of the mammal.

Another aspect of the present invention involves antisensepolynucleotide compositions that are complementary to a segment of thepolynucleotide set forth in SEQ ID NOs: 1, 3 18 and 33. Such syntheticantisense oligonucleotides are designed to bind to mRNA encoding Zcyto10polypeptides and inhibit translation of such mRNA. Such antisenseoligonucleotides are useful to inhibit expression of Zcyto10polypeptide-encoding genes in cell culture or in a subject.

The present invention also provides reagents which will find use indiagnostic applications. For example, the Zcyto10 gene, a probecomprising Zcyto10 DNA or RNA or a subsequence thereof can be used todetermine if the Zcyto10 gene is present on chromosome 1 or if amutation has occurred. Detectable chromosomal aberrations at the Zcyto10gene locus include but are not limited to aneuploidy, gene copy numberchanges, insertions, deletions, restriction site changes andrearrangements. Such aberrations can be detected using polynucleotidesof the present invention by employing molecular genetic techniques, suchas restriction fragment length polymorphism (RFLP) analysis, shorttandem repeat (STR) analysis employing PCR techniques, and other geneticlinkage analysis techniques known in the art [Sambrook et al., ibid.;Ausubel, et. al, ibid.; Marian, A. J., Chest, 108: 255-265, (1995)].

Those skilled in the art will recognize that the sequences disclosed inSEQ ID NOs: 2, 4 12, 13, 19, 20, 25, 26, 34 and 35 represent a singlealleles of the human and mouse Zcyto10 genes and polypeptides, and thatallelic variation and alternative splicing are expected to occur.Allelic variants can be cloned by probing cDNA or genomic libraries fromdifferent individuals according to standard procedures. Allelic variantsof the DNA sequence shown in SEQ ID NOs: 1, 3, 18 and 33 including thosecontaining silent mutations and those in which mutations result in aminoacid sequence changes, are within the scope of the present invention.

The sequence of Zcyto10 has 7 message instability motifs in the 3′untranslated region at positions 706, 813, 855 and 906 of SEQ ID NO:1.Treatment of cells expressing Zcyto10 with cycloheximide can alleviatethis message instability. See Shaw, G. et. al., Cell 46: 659-667 (1986).Furthermore, the AT rich 3′ untranslated region can be geneticallyaltered or removed to further promote message stability.

Use of Zcyto10 to Promote Wound Healing

The data of Example 4 shows that Zcyto10 plays a role in wound healing.Thus, Zcyto10 can be applied to a wound or a burn to promote woundhealing. Zcyto10 may be administered systemically in a dosage of from 1to 100 μg per kilogram weight of the individual. Zcyto10 may also beapplied to a wound by means of a salve or ointment which contains from 1ng to 1 mg of Zcyto10 to gram of salve or ointment. See Remington'sPharmaceutical Sciences, 18^(th) Ed., (Mack Publishing Co., Easton, Pa.,1996). Zcyto10 should be placed on a cleaned wound on a daily basisuntil the wound has healed.

Use of Zcyto10 to Increase Platelet Count

As can be seen below in Example 8, we have discovered that Zeyto10 canbe used to increase platelet count. This is especially important tocancer patients who experience thrombocytopenia due to chemotherapy orradiation therapy. The Zcyto10 can be administered therapeutically witha pharmaceutically acceptable carrier.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLE 1 Cloning of Zcyto10

The full length sequence of zcyto10x1 (the longer form) and zcyto10x2(the shorter form) was elucidated by using 3′ RACE® and submitting twofragments generated to sequencing (SEQ ID NO:10 and SEQ ID NO:11), thenartificially splicing together by computer the est sequence shown in SEQID NO:5 with the overlapping sequence from the two 3′ race fragments.

An oligo, zc15907 (SEQ ID NO: 6), was designed to the area just upstream(5′) of the putative methionine for zcyto10. Further downstream, anotheroligo, zc15906 (SEQ ID NO: 7), was designed to the area just upstream ofthe signal sequence cleavage site. These oligos were used in 3′ RACEreactions on human trachea marathon cDNA. ZC15907 was used in theprimary 3′ race reaction and zc15906 was used in the nested 3′ racereaction. The MARATHON cDNA was made using the Marathon cDNAAmplification Kit (Clontech, Palo Alto, Calif.) according to themanufacturer's instructions, starting with human trachea mRNA purchasedfrom Clontech.

The PCR reactions were run according to the manufacturer's instructionsin the Marathon cDNA Amplification Kit with some modification in thethermal cycling parameters. The cycling parameters used in the primaryPCR reaction were:

94° C. 1 min 30 sec 1×

94° C. 15 sec 68° C. 1 min 30×

72° C. 7 min 1×

The cycling parameters used in the nested PCR reaction were: 94° C. 1min 30 sec 1×, 94° C. 15 sec 68° C. 1 min 20 sec, 30×72° C. 7 min 1×.

The resulting products were run out on a 1.2% agarose gel (Gibcoagarose) and two main bands were seen, approximately 80 bp apart. Thebands were cut out and gel purified using QIAEX™ resin (Qiagen)according to the manufacturer's instructions. These fragments were thensubjected to sequencing, allowing the full length sequence of zcyto10 tobe discerned.

EXAMPLE 2 Northern Blot Analysis

Human multiple tissue blots I, II, III, and a RNA Master Dot Blot(Clontech) were probed to determine the tissue distribution of zcyto10.A 45-mer antisense oligo, SEQ ID NO:9, was designed using the estsequence (SEQ ID NO: 5 bp 100-145) and used for the probe.

15 pm of SEQ ID NO: 9 were end labeled with ³²P using T4 polynucleotidekinase (Gibco-BRL). The labeling reaction contained 2 μl 5× forwardkinase reaction buffer (Gibco-BRL), 1 ul T4 kinase, 15 pm SEQ ID NO:9, 1ul 6000 Ci/mmol ³²P gamma-ATP (Amersham) and water to 10 ul. Thereaction was incubated 30 minutes at 37° C. Unincorporated radioactivitywas removed with a NucTrap Probe Purification Column (Stratagene).Multiple tissue northerns and a human RNA Master Blot (Clontech) wereprehybridized at 50° C. three hours in 10 ml ExpressHyb (Clontech) whichcontained 1 mg of salmon sperm DNA and 0.3 mg human cot1 DNA(Gibco-BRL), both of which were boiled 3 minutes, iced 2 minutes andthen added to the ExpressHyb. Hybridization was carried out over nightat 50 C. Initial wash conditions were as follows: 2×SSC, 0.1% SDS RT for40 minutes with several wash solution changes, then 1×SSC, 0.1% SDS at64° C. (T_(m)-10) for 30 minutes. Filters were then exposed to film twodays.

Expression of zcyto10 on the northern blots revealed about a 1.2 kb bandin trachea, a faint 1.5 kb band in stomach and fainter bands of bothsizes in pancreas. The dot blots showed the presence of zcyto10 intrachea, salivary gland, placenta, testis, skin, prostate gland, adrenalgland and thyroid.

In the mouse it was found in the kidney, skeletal muscle, salivarygland, liver and skin.

EXAMPLE 3 Chromosomal Assignment and Placement of Zcyto10

Zcyto10 was mapped to chromosome 1 using the commercially availableversion of the “Stanford G3 Radiation Hybrid Mapping Panel” (ResearchGenetics, Inc., Huntsville, Ala.). The “Stanford G3 RH Panel” containsPCRable DNAs from each of 83 radiation hybrid clones of the whole humangenome, plus two control DNAs (the RM donor and the A3 recipient). Apublicly available WWW server (http://shgc-www.stanford.edu) allowschromosomal localization of markers.

For the mapping of Zcyto10 with the “Stanford G3 RH Panel”, 20 μlreactions were set up in a PCRable 96-well microtiter plate (Stratagene,La Jolla, Calif.) and used in a “RoboCycler Gradient 96” thermal cycler(Stratagene). Each of the 85 PCR reactions consisted of 2 μl 10× KlenTaqPCR reaction buffer (CLONTECH Laboratories, Inc., Palo Alto, Calif.),1.6 μl dNTPs mix (2.5 mM each, PERKIN-ELMER, Foster City, Calif.), 1 μlsense primer, SEQ ID NO: 6, 5′ ATT CCT AGC TCC TGT GGT CTC CAG 3′, 1 μlantisense primer, (SEQ ID NO: 8) 5′ TCC CAA ATT GAG TGT CTT CAG T 3′, 2μl “RediLoad” (Research Genetics, Inc., Huntsville, Ala.), 0.4 μl 50×Advantage KlenTaq Polymerase Mix (Clontech Laboratories, Inc.), 25 ng ofDNA from an individual hybrid clone or control and ×μl ddH₂O for a totalvolume of 20 μl. The reactions were overlaid with an equal amount ofmineral oil and sealed. The PCR cycler conditions were as follows: aninitial 1 cycle 5 minute denaturation at 95° C., 35 cycles of a 1 minutedenaturation at 95° C., 1 minute annealing at 66° C. and 1.5 minuteextension at 72° C., followed by a final 1 cycle extension of 7 minutesat 72° C. The reactions were separated by electrophoresis on a 2%agarose gel (Life Technologies, Gaithersburg, Md.).

The results showed linkage of Zcyto10 to the framework maker SHGC-36215with a LOD score of >10 and at a distance of 14.67cR_(—)10000 from themarker. The use of surrounding markers positions Zcyto10 in the 1q32.2region on the integrated LDB chromosome 1 map (The Genetic LocationDatabase, University of Southhampton, WWW server:http://cedar.genetics.soton.ac.uk/public_html/).

EXAMPLE 4 Use of Zcyto10 to Promote Wound Healing

Normal adult female Balb/C mice were used in the present study. Theywere housed in animal care facilities with a 12-hour light-dark cycle,given water and laboratory rodent chow ad libitum during the study. Theywere individually caged from the day of surgery.

On the day of surgery, the animals were anesthetized with ketamine(Vetalar, Aveco Inc., Ft. Dodge, Iowa) 104 mg/kg plus Xylazine (Rompun,Mobey Corp., Shawnee, Kans.) 7 mg/kg in sterile (0.2μ-filtered)phosphate buffered saline (PBS) by intraperitoneal injection. The hairon their backs was clipped and the skin depilated with NAIR®(Carter-Wallace, New York, N.Y.), then rinsed with water. 100% aloe veragel was applied to counteract the alkaline burn from the NAIR®treatment, then the animals were placed on circulating water heatingpads until the skin and surrounding fur were dry.

The animals were then anesthetized with metofane (Pittman Moore,Mundelein, N.J.) and the depilated dorsum wiped with 70% ethanol. Fourexcisions, each of 0.5-cm square were made through the skin andpanniculus carnosus over the paravertebral area at the level of thethoracic-lumbar vertebrae. The wounds and surrounding depilated skinwere covered with an adhesive, semipermeable occlusive dressing,BIOCLUSIVE® (Johnson & Johnson, Arlington, Tex.). The cut edge of theexcision was traced through the BIOCLUSIVE® onto an acetate transparencyfor later assessment of closure parameters.

Control skin and wounded skin at different time points (7 hours, 15hours and 24 hours) were processed using the Qiagen RNeasy Midi kit.Briefly, skin (control and wounded areas) were weighed and homogenizedin appropriate volume of lysis buffer (RLT). The lysates were spun toremove tissue debris and equal volume of 70% ethanol was added to thelysates; mixed well and loaded on column. The samples were spun fiveminutes and washed once with 3.8 ml of RW1 buffer, then twice with RPE(2.5 ml each). The total RNA's were eluted with RNase-free water. Theexpression level of the skin samples were measured using real time PCR(Perkin Elmer ABI Prism 7700 Sequence Detector).

The experiment was designed with a non template control, a set ofstandard and the skin samples. Mouse kidney total RNA was use for thestandard curve. Three sets of skin total RNA's (25 ng) were used in thisexperiment 7 hours (control and wounded); 15 hours (control andwounded), 24 hours (control and wounded). Each sample was done intriplicate by One Step RT-PCR on the 7700 sequence detector. Thein-house forward primer SEQ ID NO:36, reverse primer SEQ ID NO:37, andthe Perkin Elmer's TaqMan probe (ZG-7-FAM) were used in the experiment.The condition of the One Step RT-PCR was as follow: (RT step) 48° C. for30 minutes, (40 cycles PCR step) 95° C. for 10 minutes, 95° C. for 15second, 60° C. for 1 minute.

The expression level of cyto10 in the control skin samples at 7 hoursand 15 hours were comparable at 2.46 ng/ml and 2.61 ng/ml respectively.From the control skin sample at 24 hours, the expression level ofZcyto10 was zero. The expression level of cyto10 from wounded skin at 7hours was at 5.17 ng/ml (more than two fold increase compared to that ofthe control sample). The expression level of cyto10 from wounded skin at15 hours was at 14.45 ng/ml (5.5 fold increase compared to that of thecontrol sample). The expression level of cyto10 from wounded skin at 24hours was at 5.89 ng/ml. A repeat experiment also included a negativecontrol (yeast tRNA) gave the similar trend and the result of yeast tRNAwas near zero. The result suggested that the amplification was real andmouse specific.

These data suggest that Zcyto10 plays a role in the repair of woundedbecause the expression level of Zcyto10 from wounded tissue was upcompared to that of the control sample and it increased and decreasedafter time. Thus, Zcyto10 can be applied to wounds to promote woundhealing.

EXAMPLE 5 Transgenic Mice

Transgenic mice were produced which expressed Zcyto10 either under thealbumin or the metallothionine promoter. At birth, several of the micehad a shiny appearance and had limited movement. The skin of these micewas tight and wrinkled, several also had a whisker-like hair on thelower lip. The nostril and mouth areas, the extremities and the tailwere swollen.

One transgenic mouse, in which the albumin promoter was used surviveduntil day three and was severely growth retarded. There was no eardevelopment and the development of the toes was diminished. All animalswere sacrificed when they were moribund on days 1, 2 or 3. Tails andliver samples were collected and they were fixed in situ in 10% neutralformalin embedded in paraffin, and sectioned at 3 micrometers andstained with H&E. All mice with this phenotype were transgenic and hadlow to high expression of Zcyto10.

No significant changes were observed in the majority of the tissuesexcept for the skin. The skin of the zcyto10 expressing pups,particularly the those mice which had a high expression level ofZcyto10, tended to be thicker than the non-expressing pups. The stratumgranulosum in these pups appeared to be reduced in thickness as comparedto the non-expressing pups, while the stratum spinosum was thicker dueto increased cell layers and/or increased cell diameter.

In addition to the changes in the epidermis, the dermis of one mousehaving medium expression of Zcyto10 was focally moderately expanded bymucinous material.

EXAMPLE 6 Purification of Zcyto10 from a Cell Culture Medium

Zcyto10 produced by CHO cells was isolated from the cell culture mediumusing a two step method involving a cation exchange chromatography andsize exclusion chromatography.

A. Cation Exchange Chromatography Step.

Materials Used

2.2 cm diameter (D)×6 cm height (H) column (AMICON) packed with aSP-650M cation exchange resin, which is a TOYOPEARL ion exchange resinhaving covalently bonded sulfopropyl (SP) groups.

Fifteen (15) liters of culture medium from baby hamster kidney (BHK)cells which had been transfected with a Zcyto10 containing plasmid wascollected. The pH of the culture medium was adjusted to pH5 with 2N HCl.The above-described packed column was equilibrated with 50 mM sodiumacetate, NaAc, pH5.0. The culture medium was loaded onto the column atthe rate of 20 column-volumes (cv)/hr at approximately 8 ml min. Whenthe loading was done the column was washed with 10 cv of 50 mM NaAc,pH5.0. The material in the column was then eluted with 20 cv of NaClgradient in 50 mM NaAc, pH 5.0. The NaCl gradient ranged from 0 to 0.5 MNaCl. This concentrated the material in the culture medium from 15liters to 170 ml.

The resultant 170 ml harvest was further concentrated to about 5 ml witha spin 5 thousand cut-off centrifugal concentrator (Millipore, Inc.Bedford, Mass.).

B. Size Exclusion (S-100) Gel Filtration Step

Materials Used.

Column 1.6 cm (diameter)×93 cm (height)

S-100 gel (Pharmacia, Piscataway, N.J.)

The 5 ml harvest was then loaded onto the above-described columncontaining S-100 gel. The column had been equilibrated with 5× phosphatebuffered saline to bring the pH of the column to about 7.0. Zcyto10 wasisolated from the contaminants by using 1×PBS at a flow rate of 1.5ml/min. Fractions were collected at 2 ml increments. The Zcyto10polypeptide came out in fractions 52-64 at about 90 minutes after theelution had been initiated as determined by sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis which were stained with CoomassieBlue. The gel revealed one band at the predicted molecular weight ofabout 14 kDa.

EXAMPLE 7 Cloning of Murine Zcyto10

PCR primers 5′ MARATHON RACE™ (Clontech, Palo Alto, Calif.) primer setSEQ ID NO: 38 attached to MARATHON™ AP1 adapter, nested with SEQ IDNO:39 attached to AP2 MARATHON™ adapter, with 3′ MARATHON RACE™ primerset SEQ ID NO: 40 attached to MARATHON RACE™ AP1 adapter, nested withSEQ ID NO:41 attached to MARATHON RACE™ AP2 adapter and 5′ and 3′ racewas performed on mouse skin MARATHON RACE™ cDNA. Several fragments werefrom these reactions were gel purified and sequenced, allowing theelucidation of the full length coding sequence of the mouse zcyto10,plus some 5′ and 3′ UTR sequence. Two murine Zcyto10 variants werediscovered, namely SEQ ID NOs: 18 and 19 and SEQ ID NOs: 33 and 34. Theclones were amplified by PCR using primers SEQ ID NOs:42 and 43.

EXAMPLE 8 Adenovirus Administration of Zcyto10 to Normal Mice

Zcyto10 was administered by adenovirus containing the Zcyto10 gene.There were three groups of mice as described below. The adenovirus wasinjected intravenously into C57B1/6 male and female mice. All micereceived bromodeoxyuridine (BrdU) in their drinking water 3 days beforesacrifice. This allowed for detection of cell proliferation byhistologic methods. Parameters measured included weight change, completeblood counts, serum chemistries, histology, organ weights and cellproliferation by BrdU.

Experimental Design

Group 1 Zcyto10X1 (SEQ ID NO: 18)/pAC-CMV/AdV 1 × 10¹¹ particles/dose (9females, 9 males sacrificed on day 21) (2 females, 2 males sacrificed onday 11) total number = 22 mice. Group 2 null CMV/AdV control 1 × 10¹¹particles/dose (10 females, 10 males sacrificed on day 21) (2 females, 2males sacrificed on day 11) total number = 24 mice. Group 3 no treatment(5 females, 5 males) total number = 10.Results

The most striking effect was a significant increase in platelet countwhich was observed in male and female mice treated withZcyto10-adenovirus compared to empty adenovirus control. This wasaccompanied in male mice by a decrease hematocrit and increased spleenand liver weight. The thymus weight was decreased in males also. Incontrast Zcyto10-adenovirus treated female mice showed significantlyincreased white blood cell counts which were consisted primarily ofincreased lymphocyte and neutrophil counts compared to the empty viruscontrol.

These results suggest that hematopoiesis is effected by Zcyto10treatment, but except for the increased platelet count which effectedboth sexes, other effects are sex specific.

Other effects included the following.

Female glucose levels were lower in treated groups while those of themales showed no significant change.

Blood Urea Nitrogen (BUN) was higher in both male and female treatedgroups.

Female alkaline phosphatase was higher in the treated group while themales showed no significant change.

1. An isolated polynucleotide comprising a sequence of nucleotides thatencodes a polypeptide as shown in SEQ ID NO: 4 from residue 25 toresidue
 151. 2. The isolated polynucleotide according to claim 1,wherein said sequence of nucleotides encodes a polypeptide as shown inSEQ ID NO: 4 from residue 1 to residue
 151. 3. An isolatedpolynucleotide comprising a sequence of nucleotides that encodes apolypeptide as shown in SEQ ID NO:
 13. 4. The isolated polynucleotideaccording to claim 1, wherein said polynucleotide consists of a codingregion for a polypeptide as shown in SEQ ID NO: 4 from residue 25 toresidue
 151. 5. The isolated polynucleotide according to claim 2,wherein said polynucleotide consists of a coding region for apolypeptide as shown in SEQ ID NO: 4 from residue 1 to residue
 151. 6.An isolated polynucleotide that encodes a polypeptide consisting of SEQID NO:
 4. 7. An isolated polynucleotide that encodes a polypeptideconsisting of SEQ ID NO: 13.